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Structural studies of membrane proteins using transmission electron microscopy
KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology.
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Membrane proteins play important roles for living cells. They control transportation of ions, solutes, and nutrients across the membrane and catalyze metabolic reactions. Transmission electron microscopy has its advantages in convenient sample preparation, straightforward structural determination, and wide applications for diverse specimens. In this thesis, the structure of three membrane proteins are studied by this method.

Kch, a potassium channel in Escherichia coli, has a transmembrane part and a cytosolic domain. Large and well-ordered two dimensional crystals were obtained from both a functional mutant (KchM240L) and a modified protein possessing only the transmembrane part (KchTM). Both samples crystallize as two symmetry-related overlapping layers. Furthermore, the KchTM structure was reconstructed which showed that the transmembrane part of the two adjacent proteins are involved in forming the crystal contacts. Thus, the cytosolic domains of Kch in crystals are deduced to expose to the solvent and do not interact with each other.

MGST1 (microsomal glutathione transferase 1) is a detoxification enzyme. It was recombinantly over-expressed in the current study, instead of purified from rat liver as before. The crystallization condition was adjusted and isomorphic crystals were obtained. The refined model was built from a combined data set consisting of previous and new diffraction patterns. More residues at the C-terminus of the transmembrane helix 1 were assigned and the residues in the transmembrane helices 3 and 4 were remodeled. Several phospholipid molecules were observed and the ligand glutathione adopts an extended conformation in the refined model.

The structure of MelB (a sugar/sodium symporter in Escherichia coli) was determined using a refined single particle reconstruction method. This novel method is aimed for processing small or locally distorted crystals. In comparison with the previously published single particle reconstruction protocol, the current method is improved in several aspects. A more reliable reconstruction of MelB was obtained and the resolution was increased. The docking experiment indicates that MelB adopts an open conformation under the present two dimensional crystallization condition.

Electron microscopy has developed quickly recently with the help of modern instruments, techniques, and software. This method will without doubt play a more critical role in future structural biology.

 

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2015. , viii, 55 p.
Series
TRITA-STH : report, ISSN 1653-3836 ; 2015:1
National Category
Structural Biology
Identifiers
URN: urn:nbn:se:kth:diva-161721ISBN: 978-91-7595-468-4 (print)OAI: oai:DiVA.org:kth-161721DiVA: diva2:795507
Public defence
2015-04-13, Lecture hall 221, Alfred Nobels Allé 10, Flemingsberg, Huddinge, 09:00 (English)
Opponent
Supervisors
Funder
Swedish Research Council
Note

QC 20150320

Available from: 2015-03-20 Created: 2015-03-13 Last updated: 2015-09-11Bibliographically approved
List of papers
1. The projection structure of Kch, a putative potassium channel in Escherichia coli, by electron crystallography
Open this publication in new window or tab >>The projection structure of Kch, a putative potassium channel in Escherichia coli, by electron crystallography
2014 (English)In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1838, no 1, 237-243 p.Article in journal (Refereed) Published
Abstract [en]

The kch gene, the only potassium channel gene in Escherichia coil, has the property to express both full-length Kch and its cytosolic domain (RCK) due to a methionine at position 240. The RCK domains are believed to form an octameric ring structure and regulate the gating of the potassium channels after having bound certain ligands. Several different gating ring structures have been reported for the soluble RCK domains, however, these were studied isolated from their transmembrane parts. We previously reported an octameric structure of Kch in solution by electron microscopy and single particle reconstruction, composed of two tetrameric full-length proteins through RCK interaction. To exclude the effect of the detergent, we have now performed an electron crystallographic study of the full-length Kch in membrane bound form. Well-ordered two-dimensional crystals were grown in a natural phospholipid environment. A projection map merged from the fifteen best images extended to 6 angstrom resolution. The c12 two-sided plane group of the two-dimensional crystals showed that Kch crystallized as two symmetrically related overlapping layers. The arrangement suggests that the two layers of RCK domains are shifted with respect to each other and the RCK octameric gating ring of Kch does not form under the crystallization condition.

Keyword
Electron microscopy, Projection map, Potassium channel, RCK, Membrane protein
National Category
Biochemistry and Molecular Biology Biophysics
Identifiers
urn:nbn:se:kth:diva-142509 (URN)10.1016/j.bbamem.2013.09.006 (DOI)000330814000016 ()2-s2.0-84887960452 (Scopus ID)
Funder
Swedish Research Council
Note

QC 20140307

Available from: 2014-03-07 Created: 2014-03-06 Last updated: 2017-12-05Bibliographically approved
2. Free RCK Arrangement in Kch, a Putative Escherichia coli Potassium Channel, as Suggested by Electron Crystallography
Open this publication in new window or tab >>Free RCK Arrangement in Kch, a Putative Escherichia coli Potassium Channel, as Suggested by Electron Crystallography
Show others...
2015 (English)In: Structure, ISSN 0969-2126, E-ISSN 1878-4186, Vol. 23, no 1, 199-205 p.Article in journal (Refereed) Published
Abstract [en]

The ligand-gated potassium channels are stimulated by various kinds of messengers. Previous studies showed that ligand-gated potassium channels containing RCK domains (the regulator of the conductance of potassium ion) form a dimer of tetramer structure through the RCK octameric gating ring in the presence of detergent. Here, we have analyzed the structure of Kch, a channel of this type from Escherichia coli, in a lipid environment using electron crystallography. By combining information from the 3D map of the transmembrane part of the protein and docking of an atomic model of a potassium channel, we conclude that the RCK domains face the solution and that an RCK octameric gating ring arrangement does not form under our crystallization condition. Our findings may be applied to other potassium channels that have an RCK gating ring arrangement.

National Category
Structural Biology
Identifiers
urn:nbn:se:kth:diva-160349 (URN)10.1016/j.str.2014.10.018 (DOI)000347469500024 ()2-s2.0-84920999760 (Scopus ID)
Funder
Swedish Research Council
Note

QC 20150623

Available from: 2015-02-18 Created: 2015-02-18 Last updated: 2017-12-04Bibliographically approved
3. A refined atomic model for microsomal glutathione transferase 1 from electron crystallography
Open this publication in new window or tab >>A refined atomic model for microsomal glutathione transferase 1 from electron crystallography
Show others...
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Microsomal glutathione transferase 1 (MGST1) is a detoxification enzyme belonging to the Membrane Associated Proteins in Eicosanoid and Glutathione Metabolism (MAPEG) superfamily. Here we have used electron crystallography of two-dimensional (2D) crystals in order to determine an atomic model of rat MGST1 in a lipid environment. The 2D crystals were of the p6 two-sided plane group symmetry. For the refinement, information to 3.5 Å resolution from 225 electron diffraction patterns recorded from specimens at tilt angles up to 66° was used. The model comprises 123 of the 155 amino acid residues, two structured phospholipid molecules, two hydrocarbon chains, and one glutathione (GSH) molecule. Interactions between subunits form trimers centered on the crystallographic three-fold axes of the unit cell. The GSH substrate binds in an extended conformation at the interface between two subunits of the trimer. The location of GSH is supported by mutagenesis data in vitro.

National Category
Structural Biology
Identifiers
urn:nbn:se:kth:diva-161719 (URN)
Funder
Swedish Research Council
Note

QS 2015

Available from: 2015-03-13 Created: 2015-03-13 Last updated: 2015-03-20Bibliographically approved
4. A Refined Single-Particle Reconstruction Procedure to Process Two-Dimensional Crystal Images from Transmission Electron Microscopy
Open this publication in new window or tab >>A Refined Single-Particle Reconstruction Procedure to Process Two-Dimensional Crystal Images from Transmission Electron Microscopy
Show others...
2015 (English)In: Microscopy and Microanalysis, ISSN 1431-9276, E-ISSN 1435-8115, Vol. 21, no 4, 876-85 p.Article in journal (Refereed) Published
Abstract [en]

Single-particle reconstruction (SPR) and electron crystallography (EC), two major applications in electron microscopy, can be used to determine the structure of membrane proteins. The three-dimensional (3D) map is obtained from separated particles in conventional SPR, but from periodic unit cells in EC. Here, we report a refined SPR procedure for processing 2D crystal images. The method is applied to 2D crystals of melibiose permease, a secondary transporter in Escherichia coli. The current procedure is improved from our previously published one in several aspects. The "gold standard Fourier shell correlation" resolution of our final reconstruction reaches 13 A, which is significantly better than the previously obtained 17 A resolution. The choices of different refinement parameters for reconstruction are discussed. Our refined SPR procedure could be applied to determine the structure of other membrane proteins in small or locally distorted 2D crystals, which are not ideal for EC.

Place, publisher, year, edition, pages
Cambridge University Press, 2015
Keyword
cryo-EM, electron crystallography, melibiose permease, membrane proteins, single-particle reconstruction
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-171876 (URN)10.1017/S1431927615000616 (DOI)000358834600009 ()25990985 (PubMedID)2-s2.0-84938421884 (Scopus ID)
Funder
Swedish Research Council
Note

QC 20150817

Available from: 2015-08-17 Created: 2015-08-10 Last updated: 2017-12-04Bibliographically approved

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